Functional and phenotypic properties of peripheral T cells anergized by autologous CD3⁺ depleted bone marrow cells

We have previously demonstrated that bone marrow cells (BMC) inhibit the generation of autologous Epstein-Barr virus (EBV) -specific cytotoxic T lymphocytes (CTL). It was also observed that CD3⁺ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. In the present study, we examined these BMC induced regulator CD3⁺ T cells with respect to phenotype, function, and T-cell activation pathways. We also questioned if the CD3⁺ regulatory cell function is mediated by their direct effect on peripheral T cells or on the ability of antigen presenting cells (APC) to stimulate peripheral T cells. To answer this, CD3⁺ cells from peripheral blood lymphocytes (PBL) were cultured with either CD3-depleted BMC or with CD3-depleted PBL. The CD3⁺ cells were then isolated with immunomagnetic beads, designated as T_BM and T_PBL, and were compared in functional studies. There was an increase in the expression of CD25 on T_BM cells. The T_BM cells also expressed less CD122 and a decreased number of CD3 molecules per cell. Both T_BM and T_PBL cell populations responded to mitogen (PHA) to the same magnitude. However, when stimulated through the CD3 complex with anti-CD3 monoclonal antibody (mAb), the T_BM cells had a significantly decreased response than did T_PBL. The addition of IL-2 to these latter cultures augmented, but could not fully restore, the response. Additionally, stimulation of T_BM cells with allogeneic cells failed to produce cytotoxic T cells. These "anergized" T_BM and "nonanergized" (control) T_PBL cells were added as third-party cells to a CTL generating culture of autologous PBL stimulated with allogeneic cells. The T_BM cells exhibited suppressor function and inhibited the generation of CTL, in contrast with T_PBL. The effect of T_BM cells on direct and indirect antigen presentation pathways demonstrated that T_BM primarily effected indirect, but not direct, alloantigen presentation. To further explore the cytoplasmic T-cell activation events that occurred after the coculture of the PBL T cells with BMC, the levels of zeta-associated protein 70 (ZAP70) and extracellular receptor-activated kinase (ERK) were determined. There was a decrease in ZAP70 levels in the T_BM, which correlated with its reduced expression of cell surface CD3 and the attenuated response to anti-CD3 mAb activation. However, the activity of ERK was equally expressed by T_BM and T_PBL. It, therefore, appears that the culturing of peripheral T cells with (non-T) BMC anergizes these cells (which become refractory to stimulation through the T-cell receptors), and induces immune suppressor function. These in vitro observations may provide a mechanism by which infused donor BMC serve to downregulate T-cell immunity.